1. Installation

System requirements

PSFj runs on Windows 7, Linux, and MacOS 10.9 and requires Java 7

Recommended minimum hardware configuration is:

• 64-bits OS and JAVA version
• Dual-core processor
• 4GB of RAM*

* the amount of RAM required for an analysis is about twice the size of the analyzed data. In other words, to analyze 4GB of data, PSFj requires 8GB of RAM.

Important note : On 32-bits systems PSFj will use a maximum of 2GB of memory regardless of how much memory the computer is equipped with.

Run PSFj

To run PSFj, extract the zip-file ( psfj_latest.zip for Windows and Linux, or
psfj_macos_latest.zip for MacOS). It will extract a folder “PSFj” containing executables (psfj.exe for Windows, psfj for Linux, and PSFj for MacOS) and a library “psfj__lib” folder.

Note : PSFj runs only if the “psfj__lib” folder is in the same folder as the executables. For convenience you can generate a shortcut and place it in a place of your choice, e.g. desktop.

Testing the installation

Download a sample stack from the publication here. The archives contain .ini files that provide the settings: information about the used microscope, bead size etc.. These are automatically read by PSFj. Run PSFj, for instructions, see section 3 (Running PSFj) below.

Compare results with the ones in the publication.

2. Collect imaging parameters

Before analyzing your own data, you need to identify a number of imaging parameters used to acquired your data. Those are:

• The magnification (M) and the numerical aperture (NA) of your objective lens (e.g. 60x, NA=1.40 – given on the body of the objective)
• The pixel size of your camera (e.g. 6.5 µm x 6.5 µm, provided in the camera data sheet (use a web search engine to find this info based on the name of your camera)
• The refractive index (n) of the used immersion fluid (e.g. n = 1 for air, n = 1.33 for water, n = 1.47 for glycerol, n = 1.52 for oil – check the oil container)
• The diameter of the used fluorescent beads (e.g. 170 nm) and the center wavelength(s) of the emission filter used for imaging (e.g. 520 nm).

3. Fluorescent bead slide and image acquisition

Preparation of the bead slide

For direct extraction of PSFs from an image stack of fluorescent beads (use multi-spectral beads if you intend to measure chromatic aberrations), the beads should ideally be as small as possible. However the smaller the beads the lower the signal-to-noise ratio and the more difficult the quantification becomes. We thus recommend to use beads with a diameter of 100 – 200 nm. PSFj will automatically correct for the bead size. Also, check that your beads are essentially aggregate free. If necessary, try to disperse these aggregates using a water bath sonicator.

The beads should also exhibit a narrow size distribution, i.e., a small coefficient of variation of their diameter (check the specification of the manufacturer).

For preparation of the slide, follow the manufacturer protocol which usually includes the following steps.

1. Dilute the bead solution to a suitable concentration (around 10^9 beads per ml)
2. add a small amount (around 5 µl) on top of a coverslip and let it dry. Consider that some objective lenses require coverslips of a specific thickness.
3. cover the beads with a small amount (e.g. 5 µl) of mounting medium and place the coverslip on a glass slide. Make sure that the coverslip is planar with respect to the glass slide: apply even pressure when mounting the coverslip, and avoid dust particles between coverslip and glass slide. Seal e.g. with nail polish.

Image acquisition

Record a stack of fluorescence images of your beads with the best focus set (manually) to the center of the stack. The stack should cover a range of about 2 to 3 times the expected z resolution (2λn/NA²) to both sides of (above and below) the best focus with a spacing of about (2λn/NA²)/10.

PSFj assumes, that the camera pixel size is small enough to provide sufficient sampling (at least Nyquist) of the lateral extent of the PSF, i.e., is smaller than (0.61λM/NA)/2. If this is not the case it might be possible to increase the overall magnification (e.g. 1.5x) through the use of a different tube lens as is provided by most microscopes. In addition, camera binning should be generally avoided.

 

4. Preparing image stack for PSFj

PSFj requires a multi-page tif as input. Converting stacks of different formats or single images into a multi-page tif can be done using ImageJ or Fiji .

 

5. Interpretation of Results, repetition of measurements

PSFj outputs heat maps that allow you to visually inspect the performance of your microscope and objective lens. Heat maps can be generated in absolute scale (nm) or normalized to the theoretical performance of the system (in the range of 0.5 to 2 times the theoretical performance).

For interpretation of the results, read the supplementary information of the publication.

(more hints and examples will be provided here at a later time point)